The present invention relates to new substituted imidazotriazinones, processes for their preparation, and their use for the production of medicaments, in particular for improving perception, concentration power, learning power and/or memory power.
Phosphodiesterases (PDEs) play an essential role in the regulation of the intracellular cGMP and cAMP levels. Of the previously described phosphodiesterase isoenzyme groups PDE 1 to PDE 10 (Beavo and Reifsnyder Trends in Pharmacol. Sci. 1990, 11, 150-155; Sonderling and Beavo Curr. Opin. Cell Biol. 2000, 12, 174-179), the PDEs 1, 2, 5, 9 and 10 are mainly responsible for the metabolism of cGMP. On account of the varying distribution of these cGMP-metabolizing PDEs in the tissue, selective inhibitors should raise the cGMP levels in the corresponding tissue, depending on the tissue distribution of the appropriate isoenzyme.
The particular feature of PDE 2 lies in its positive cooperative kinetics with respect to the substrate cGMP. It was postulated that small amounts of cGMP bind to the so-called cGMP-binding domain and thereby bring about activation of the enzyme. By this means, the affinity of the catalytic domain to cGMP and cAMP is also increased (Martins et al. J. Biol. Chem. 1982, 257, 1973-1979). Therefore PDE 2 can hydrolyse and thereby also control both second messenger systems by means of small amounts of cGMP.
PDE 2 has been isolated from various tissues, for example from heart, adrenal gland, liver, platelets and in particular brain. In the brain, PDE 2 mRNA is expressed strongly in the cortex, the basal ganglia and in the hippocampus (Sonnenburg et al. Biol. Chem. 1991, 266, 17655-17661). The sequence of the human isoform PDE 2A3 was reported by Rosman et al. Gene 1997, 191, 89-95. Of the tissues investigated, the expression of PDE 2A was demonstrated strongly therein in the brain and heart and more weakly in liver, skeletal muscle, kidney and pancreas.
U.S. Pat. No. 4,278,673 discloses imidazopyrimidinones having cAMP PDE-inhibitory action for the treatment of asthma and bronchitis.
WO-A-99/67244 and WO-A-99/24433 disclose 7-alkyl-2-phenyl-imidazotriazinones having PDE 5-inhibiting action for the treatment of vascular diseases.
EP-A-0 771 799, WO-A-98/40384 and WO-A-00/12504 describe purinone, allopurinol and triazolopyrimidinone derivatives, their inhibitory action on cGMP-metabolizing PDEs and their suitability for the treatment of vascular diseases.
The present invention relates to compounds of the general formula (I), 
in which
R1 denotes phenyl, naphthyl, quinolinyl or isoquinolinyl, each of which can be substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy, halogen, cyano, xe2x80x94NHCOR8, xe2x80x94NHSO2R9, xe2x80x94SO2NR10R11, xe2x80x94SO2R12, and xe2x80x94NR13R14,
in which
R8, R10, R11, R13 and R14 independently of one another are hydrogen or (C1-C4)-alkyl, and
R9 and R12 independently of one another are (C1-C4)-alkyl, or
R10 and R11 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical, or
R13 and R14 together with the adjacent nitrogen atom form an azetidin-1-yl, pyrrol-1-yl, piperid-1-yl, azepin-1-yl, 4-methyl-piperazin-1-yl or morpholin-1-yl radical,
R2 and R3 independently of one another denote hydrogen or fluorine,
R4 denotes (C1-C4)-alkyl,
R5 denotes (C1-C3)-alkyl,
R6 denotes hydrogen or methyl,
R7 denotes phenyl, thiophenyl, furanyl, each of which can be substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy, halogen and cyano, or (C5-C8)-cycloalkyl,
L denotes carbonyl or hydroxymethanediyl, and
M denotes (C2-C5)-alkanediyl, (C2-C5)-alkenediyl or (C2-C5)-alkinediyl,
and their physiologically tolerable salts.
(C1-C4)-Alkyl and (C1-C3)-alkyl in the context of the invention represent a straight-chain or branched alkyl radical having 1 to 4 and 1 to 3 carbon atoms respectively. Examples which may be mentioned are: methyl, ethyl, n-propyl, isopropyl, i-, s-, t-butyl. Methyl and ethyl are preferred.
(C2-C5)-Alkanediyl in the context of the invention represents a straight-chain or branched alkanediyl radical having 2 to 5 carbon atoms. Examples which may be mentioned are ethylene, propane-1,3-diyl, propane-1,2-diyl, propane-2,2-diyl, butane-1,3-diyl, butane-2,4-diyl, pentane-2,4-diyl. A straight-chain (C2-C5)-alkane-1,xcfx89-diyl radical is preferred. Examples which may be mentioned are ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl. Propane-1,3-diyl and butane-1,4-diyl are particularly preferred.
(C2-C5)-Alkenediyl in the context of the invention represents a straight-chain or branched alkenediyl radical having 2 to 5 carbon atoms. Examples which may be mentioned are ethene-1,2-diyl, ethene-1,1-diyl, propene-1,1-diyl, propene-1,2-diyl, prop-2-ene-1,3-diyl, propene-3,3-diyl, propene-2,3-diyl, but-2-ene-1,4-diyl, pent-2-ene-1,4-diyl. A straight-chain (C2-C5)-alkene-1,xcfx89-diyl radical is preferred. Examples which may be mentioned are ethene-1,2-diyl, prop-2-ene-1,3-diyl, but-2-ene-1,4-diyl, but-3-ene-1,4-diyl, pent-2-ene-1,5-diyl, pent-4-ene-1,5-diyl. Prop-2-ene-1,3-diyl, but-2-ene-1,4-diyl and but-3-ene-1,4-diyl are particularly preferred.
(C2-C5)-Alkinediyl in the context of the invention represents a straight-chain or branched alkinediyl radical having 2 to 5 carbon atoms. Examples which may be mentioned are ethine-1,2-diyl, ethine-1,1-diyl, prop-2-ine-1,3-diyl, prop-2-ine-1,1-diyl, but-2-ine-1,4-diyl, pent-2-ine-1,4-diyl. A straight-chain (C2-C5)-alkene-1,xcfx89-diyl radical is preferred. Examples which may be mentioned are ethine-1,2-diyl, prop-2-ine-1,3-diyl, but-2-ine-1,4-diyl, but-3-ine-1,4-diyl, pent-2-ine-1,5-diyl, pent-4-ine-1,5-diyl. Prop-2-ine-1,3-diyl, but-2-ine-1,4-diyl and but-3-ine-1,4-diyl are particularly preferred.
(C1-C4)-Alkoxy in the context of the invention represents a straight-chain or branched alkoxy radical having 1 to 4 carbon atoms. Examples which may be mentioned are: methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-pentoxy and n-hexoxy. Methoxy and ethoxy are preferred.
(C5-C8)-Cycloalkyl in the context of the invention represents cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. The following may preferably be mentioned are: cyclopentyl, cyclohexyl or cycloheptyl.
Halogen in the context of the invention in general represents fluorine, chlorine, bromine and iodine. Fluorine, chlorine and bromine are preferred. Fluorine and chlorine are particularly preferred.
Preferred salts in the context of the invention are physiologically acceptable salts of the compounds according to the invention.
Physiologically acceptable salts of the compounds according to the invention can be acid addition salts of the substances according to the invention with mineral acids, carboxylic acids or sulphonic acids. Particularly preferred salts are, for example, those with hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, toluenesulphonic acid, benzenesulphonic acid, naphthalenedisulphonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
Salts which may be mentioned are, however, also salts with customary bases, such as, for example, alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts) or ammonium salts derived from ammonia or organic amines such as, for example, diethylamine, triethylamine, ethyldiisopropylamine, procaine, dibenzylamine, N-methylmorpholine, dihydroabietylamine, 1-ephenamine or methyl-piperidine.
The compounds according to the invention can exist in stereoisomeric forms, which either behave as image and mirror image (enantiomers), or which do not behave as image and mirror image (diastereomers). The invention relates both to the enantiomers and diastereomers or their respective mixtures. Like the diastereomers, the racemic forms can be separated into the stereoisomerically uniform constituents in a known manner.
Preferred compounds of the general formula (I) are those where R1 denotes phenyl whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and xe2x80x94SO2NR10R11, and R2, R3, R4, R5, R6, R7, R10, R11, L and M have the meaning indicated above.
The meta and para positions of the phenyl ring are understood as meaning those positions which are meta or para to the CR2R3 group. These positions can be illustrated by the following structural formula (Ic): 
Particularly preferred compounds of the general formula (Ic) are those in which the para and one meta position of the phenyl radical are substituted, and the second meta position is unsubstituted.
Likewise, preferred compounds of the general formula (I) are those where R7 denotes phenyl and R1, R2, R3, R4, R5, R6, L and M have the meaning indicated above.
Very particularly preferred are compounds of the general formula (I),
where
R1 denotes phenyl, whose meta and/or para positions are substituted up to three times identically or differently by radicals selected from the group consisting of (C1-C4)-alkyl, (C1-C4)-alkoxy and xe2x80x94SO2NR10R11, naphthyl or quinolinyl,
in which R10 and R11 independently of one another are hydrogen or (C1-C4)-alkyl,
R1 and R2 denote hydrogen,
R4 denotes methyl or ethyl,
R5 denotes methyl,
R6 denotes hydrogen or methyl,
L denotes carbonyl or hydroxymethanediyl, and
M denotes straight-chain (C2-C5)-alkane-1,xcfx89diyl, straight-chain (C2-C5)-alkene-1,xcfx89-diyl or straight-chain (C2-C5)-alkine-1,xcfx89-diyl.
A further aspect of the invention relates to a new preparation process for the compounds of the general formula (I), in which R1, R2, R3, R4, R5, R6, R7, L and M have the meaning indicated above,
where
[A] a compound of the general formula (IIa), 
in which R1, R2, R3, R4, R5, R6, R7 and M have the meaning indicated in Claim 1,
xe2x80x83is reacted under suitable condensation conditions to give a compound of the general formula (Ia), 
in which R1, R2, R3, R4, R5, R6, R7 and M have the meaning, indicated in Claim 1,
xe2x80x83and then, if appropriate,
[B] is reduced under suitable conditions to give a compound of the general formula (Ib) 
in which R1, R2, R3, R4, R5, R6, R7 and M have the meaning indicated in Claim 1.
The condensation according to reaction step [A] can be carried out by heating the compounds of the general formula (IIa) in the absence of a solvent or in the presence of an inert solvent, in particular of a solvent of the type which forms an azeotropic mixture with water, such as, for example, toluene or xylene, if appropriate in the presence of an acid catalyst and/or of a dehydrating agent. A suitable acid catalyst is, for example, hydrogen chloride and a dehydrating agent which can be used is, for example, acetyl chloride, phosphorus pentoxide or phosphoryl chloride. The condensation is preferably carried out in an inert solvent in the presence of 1-10, preferably 3-7, equivalents of phosphoryl chloride (cf. Chem. Ind. 1983, 331-335).
Suitable inert solvents for the condensation are the customary organic solvents which do not change under the reaction conditions. These include, for example, ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or hydrocarbons such as benzene, toluene, xylene, hexane, cyclohexane or petroleum fractions, or halogenohydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, trichloroethylene or chlorobenzene, or ethyl acetate, dimethylformamide, hexamethylphosphoramide, acetonitrile, acetone, dimethoxyethane or pyridine. It is likewise possible to use mixtures of the solvents mentioned. 1,2-Dichloroethane is preferred.
The reaction temperature can be varied within a relatively wide range. In general, the reaction is carried out in a range from xe2x88x9220xc2x0 C. to 200xc2x0 C., preferably from xe2x88x9220xc2x0 C. to 90xc2x0 C.
The process steps according to the invention are in general carried out at normal pressure. However, it is also possible to work at elevated pressure or at reduced pressure (e.g. in a range from 0.5 to 5 bar).
The reduction according to reaction step [B] can be carried out according to customary methods.
The reductions are in general carried out using hydrides or using boranes, diboranes or their complex compounds in inert solvents.
The reductions can also be carried out by means of hydrogen in water or in inert solvents such as alcohols, ethers or halogenohydrocarbons, or their mixtures, using catalysts such as Raney nickel, palladium, palladium on active carbon or platinum.
Preferably, the reductions are carried out using hydrides, such as complex borohydrides or aluminium hydrides. Particularly preferably, sodium borohydride, lithium borohydride, sodium cyanoborohydride, lithium aluminium hydride, sodium bis(2-methoxyethoxy)aluminium hydride or borane/tetrahydrofuran are employed here.
Suitable solvents here for the reduction are all solvents which do not change under the reaction conditions. These preferably include alcohols such as methanol, ethanol, n-propanol or isopropanol, or ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, diethylene glycol dimethyl ether or amides such as hexamethylphosphoramide or dimethylformamide, or acetic acid. It is likewise possible to use mixtures of the solvents mentioned.
The reduction is in general carried out in a temperature range from xe2x88x9250xc2x0 C. up to the respective boiling point of the solvent, preferably from xe2x88x9220xc2x0 C. to +90xc2x0 C., particularly preferably from xe2x88x925xc2x0 C. to 30xc2x0 C.
If necessary, the compounds of the general formula (I) can be separated into the pure diastereomers and/or pure enantiomers. For example, chromatographic separation under normal-, medium- or high-pressure conditions on stationary phases such as, for example, silica gel or reversed phase-modified silica gels or chirally modified silica gels is suitable for this purpose. This is preferably carried out by the high-performance liquid chromatography (=HPLC) method using chiral stationary silica gel phases. Chiral polyamide/silica gel phases based on the monomers N-methacryloyl-L-leucine-d-menthylamide or N-methacryloyl-L-leucine-1-menthylamide are particularly suitable for the separation of the racemates (cf. EP-A-0 379 917).
It can also prove favourable to employ diastereomerically and/or enantiomerically pure compounds of the general formula (IIa) in reaction step [A] and/or to separate the compounds of the general formula (Ia) into the pure diastereomers and/or enantiomers, if appropriate, before carrying out reaction step [B].
It is likewise possible to carry out the reduction [B] diastereoselectively. For this purpose, the reduction is expediently carried out using hydrides, such as complex borohydrides or aluminium hydrides and also boranes in the presence of metal salts. Particularly preferred metal salts are those whose cations are capable of bidentate coordination, such as, for example, metals of the main groups IIa and IIIa or metals of the subgroups including the lanthanoids. Salts of Zn, Mn, Mg or Ca are particularly preferred. Anions which can be used are, for example, halides or acetates. The reaction is expediently carried out in an alcohol or a mixture of an alcohol and a further inert solvent. Mixtures of methanol or ethanol and dichloromethane are preferred. The reduction is in general carried out in a temperature range from xe2x88x9250xc2x0 C. up to the respective boiling point of the solvent, preferably from xe2x88x9220xc2x0 C. to +90xc2x0 C., particularly preferably from xe2x88x925xc2x0 C. to 30xc2x0 C.
The reduction is carried out, inter alia, using 1 to 20 equivalents of the reducing agent in the presence of 0.1 to 10 equivalents of metal salt. In a preferred embodiment, 0.2 to 3 equivalents of metal salt are used. Preferred reducing agents are, for example, sodium borohydride, lithium borohydride, sodium cyanoborohydride or zinc borohydride.
The intermediates of the general formula (II) are new.
A further aspect of the present invention therefore relates to the new compounds of the general formula (II), 
in which R1, R2, R3, R4, R5, R6, R7, L and M have the meaning indicated above,
and their salts.
The compounds of the general formula (IIa) can be prepared, for example, according to known methods by the oxidation of corresponding compounds of the general formula (IIb), 
in which R1, R2, R3, R4, R5, R6, R7 and M have the meaning indicated in Claim 1, for example by Swern oxidation or Collins oxidation (for further oxidation methods also see March, J. M., xe2x80x9cAdvanced Organic Chemistryxe2x80x9d, 3rd Edition, John Wiley, New York, 1985, pp. 1057-1060 and literature cited therein).
The preparation of the compounds of the general formula (II) can be illustrated by way of example by the following synthesis scheme: 
The compounds of the general formulae (III), (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI) are known or can be prepared by known processes.
According to this reaction scheme, the aminomethyltriazinones (IIIb) are condensed with the carboxylic acids (IV) under the conditions customary for the formation of amide bonds using a dehydrating reagent in an inert solvent, if appropriate in the presence of a base.
Suitable dehydrating reagents are carbodiimides, such as, for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide (DCC) or N-(3-dimethylaminopropyl)-Nxe2x80x2-ethylcarbodiimide hydrochloride (EDC) or carbonyl compounds such as carbonyldiimidazole or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulphonate or propanephosphonic anhydride or isobutyl chloroformate or benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate or diphenyl phosphoramidate or methanesulphonyl chloride, if appropriate in the presence of bases such as triethylamine, N-ethylmorpholine, N-methylmorpholine or N-methylpiperidine, and if appropriate in the presence of a catalyst such as N-hydroxysuccinimide or N-hydroxybenzotriazole (HOBT). The condensation with EDC is preferably carried out in the presence of NMM and HOBT.
Suitable solvents are the customary inert solvents described above. Dichloromethane is preferred.
The aminomethyltriazinones (IIIb) are obtainable by deprotection of the corresponding N-protected aminomethyltriazinones (IIIa), which in turn are accessible via cyclo-condensation of the corresponding amidrazones (Vb) and xcex1-keto esters (VI).
Suitable amino protective groups for the intermediates (IIIa), (VI) and (VIII) are, for example, acyl radicals, in particular the acetyl group. These groups can be cleaved into the N-protected aminomethyltriazinones (IIIa) under acidic conditions, for example by heating in hydrochloric acid.
The cyclocondensation to give the N-protected aminomethyltriazinones (IIIa) can be brought about by heating the individual components, the amidrazones (Vb) and xcex1-keto esters (VI), in an alcoholic solvent, preferably by heating to reflux in ethanol.
The amidrazones (Vb) can be prepared by reaction of the corresponding amidines (Va) with, for example, hydrazine hydrate and are either isolated or employed in situ in the following reaction. The amidines (Va) are accessible from the corresponding nitrites (VII) according to customary methods, for example by reaction of the nitrites (VII) with ammonium chloride and a solution of trimethylaluminium in hexane firstly in a temperature range from xe2x88x9220xc2x0 C. to room temperature, preferably at 0xc2x0 C. and then at 60 to 100xc2x0 C., preferably 70 to 90xc2x0 C., and preferably at normal pressure.
The nitriles (VII) are known or can be prepared according to customary methods. For example, aryl-difluoro-acetonitriles can be prepared from arylacetonitriles or aryloxoacetonitriles (cf. J. Org. Chem. 1998, 63, 8052-8057 or J. Fluorine Chem. 1996, 76, 15-20).
The xcex1-keto esters (VI) can be prepared from the corresponding N-protected xcex1-amino acids (VIII), for example by reaction with ethyl oxalyl chloride.
The carboxylic acids (IV) are accessible by alkylation of the corresponding xcex2-keto esters (IX) with the electrophiles (X) and if appropriate (XI), followed by ester hydrolysis and, if appropriate, reduction of the xcex2-carbonyl function.
For alkylation, the xcex2-ketoester (IX) is deprotonated for example using a base, preferably a hydride such as sodium hydride, in an inert solvent such as, for example, tetrahydrofuran in a temperature range from preferably 0xc2x0 C. to room temperature and, after isolation or in situ, treated with a solution of the electrophile (X) or (XI) in, preferably, 1,3-dimethyltetrahydro-2-(1H)-pyrimidone with addition of a catalytic amount of potassium iodide. If R6 is not hydrogen, the alkylation can be repeated using a second electrophile after the monoalkylation product has optionally been isolated. The leaving group Y in the electrophile (X) or (XI) is preferably a halogen, particularly preferably bromine.
The xcex2-carbonyl function can be reduced according to the conditions described above for reaction step [B].
The hydrolysis of the ester to the carboxylic acid (IV) is carried out according to customary conditions, in the case of the methyl or ethyl ester preferably using sodium or potassium hydroxide solution.
Substituents, for example in R1, can be introduced via the starting materials, such as, for example, via the nitrile (VII), but can also be introduced or modified in a later process stage.
Thus the substituent xe2x80x94SO2NR10R11, for example, can be introduced into R1 by chlorosulphonating an appropriate N-protected aminomethyltriazinone (IIIa) with chlorosulphonic acid and then further reacting it with an appropriate amine HNR10R11 to give the corresponding sulphonamide.
This can be illustrated by the following reaction scheme: 
The compounds according to the invention show an unforeseeable, valuable spectrum of pharmacological action: they preferably inhibit PDE 2, and/or exhibit a favourable pharmacokinetic profile.
The inhibition of PDE 2 leads to a differentiated increase in cGMP. The differentiating action is additionally determined by the distribution of the isoenzymes in the tissue.
The compounds according to the invention moreover intensify the action of substances, such as, for example, EDRF (endothelium-derived relaxing factor) and ANP (atrial natriuretic peptide), which increase the cGMP level.
Because of their selective PDE 2 inhibition, the compounds according to the invention are particularly suitable for improving perception, concentration power, learning power or memory power, in particular after cognitive disorders, such as occur, for example, in situations/illnesses/syndromes such as mild cognitive impairment, age-associated learning and memory disorders, age-associated memory losses, vascular dementia, craniocerebral trauma, stroke, dementia which occurs after strokes (post-stroke dementia), post-traumatic craniocerebral trauma, general concentration disorders, concentration disorders in children with learning and memory problems, Alzheimer""s disease, vascular dementia, dementia with Lewy bodies, dementia with degeneration of the frontal lobes including Pick""s disease, Parkinson""s disease, progressive nuclear palsy, dementia with corticobasal degeneration, amyolateral sclerosis (ALS), Huntington""s disease, multiple sclerosis, thalamic degeneration, Creutzfeld-Jacob dementia, HIV dementia, schizophrenia with dementia or Korsakoff psychosis.
The compounds according to the invention are generally suitable for the treatment and/or prophylaxis of dementia.
The active compound can act systemically and/or locally. For this purpose, it can be administered in a suitable manner, such as, for example, orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, transdermally, conjunctivally, otically or as an implant.
For these administration routes, the active compound can be administered in suitable administration forms.
For oral administration, known administration forms releasing the active compound rapidly and/or in modified form are suitable, such as, for example, tablets (non-coated and coated tablets, e.g. enteric coatings), capsules, sugar-coated tablets, granules, pellets, powders, emulsions, suspensions and solutions.
The parenteral administration can take place with circumvention of an absorption step (intravenous, intraarterial, intracardiac, intraspinal or intralumbar) or with involvement of an absorption (intramuscular, subcutaneous, intracutaneous, percutaneous, or intraperitoneal). For parenteral administration, suitable administration forms are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilizates and sterile powders.
For the other administration routes, for example, inhalation pharmaceutical forms (inter alia powder inhalers, nebulizers), nasal drops/solutions, sprays; tablets or capsules to be applied lingually, sublingually or buccally, suppositories, ear and eye preparations, vaginal capsules, aqueous suspensions (lotions, shake lotions), lipphilic suspensions, ointments, creams, milk, pastes, dusting powder or implants are suitable.
The active compounds can be converted into the administration forms mentioned in a known manner. This takes place using inert non-toxic, pharmaceutically suitable excipients. These include, inter alia, vehicles (e.g. microcrystalline cellulose), solvents (e.g. liquid polyethylene glycols), emulsifiers (e.g. sodium dodecylsulphate), dispersants (e.g. polyvinylpyrrolidone), synthetic and natural biopolymers (e.g. albumin), stabilizers (e.g. antioxidants such as ascorbic acid), colourants (e.g. inorganic pigments such as iron oxides) or taste and/or odour corrigents.
In general, it has proved advantageous in the case of parenteral administration to administer amounts of approximately 0.001 to 30 mg/kg, preferably approximately 0.01 to 10 mg/kg, of body weight to achieve effective results. In the case of oral administration, the amount is approximately 0.01 to 100 mg/kg, preferably approximately 0.1 to 30 mg/kg, of body weight.
In spite of this, it may be necessary, if appropriate, to deviate from the amounts mentioned, namely depending on the body weight, route of application, individual behaviour towards the active compound, manner of preparation and time or interval at which administration takes place.
Measurement of the PDE Inhibition
The cGMP-stimulable PDE (PDE 2), the cGMP-inhibitable PDE (PDE 3) and the cAMP-specific PDE (PDE 4) were isolated either from porcine or bovine heart myocardium. The Ca2+ calmodulin-stimulable PDE 1 was isolated from porcine aorta, porcine brain or preferably from bovine aorta. The cGMP-specific PDE (PDE 5) was preferably obtained from porcine small intestine, porcine aorta, human blood platelets and preferably from bovine aorta. Purification was carried out by anion exchange chromatography on mono QR Pharmacia essentially according to the method of Hoey, M; Houslay, M. D., Biochem. Pharmacol. 1990, 40, 193-202 and Lugman et al. Biochem. Pharmacol. 1986, 35, 1743-1751.
The enzyme activity was determined in a test batch of 100 xcexcl in 20 mM tris/HCl buffer pH 7.5 which contains 5 mM MgCl2, 0.1 mg/ml of bovine serum albumin and either 800 Bq of [3H]-cAMP or [3H]-cGMP. The final concentration of the corresponding nucleotides is 10xe2x88x926 mol/l. The reaction is started by addition of the enzyme and the amount of enzyme is proportioned such that about 50% of the substrate are reacted during the incubation time of 30 min. In order to test the cGMP-stimulable PDE 2, [3H]-cAMP is used as a substrate and 10xe2x88x926 mol/l of non-labelled cGMP is added to the batch. In order to test the Ca calmodulin-dependent PDE 1, additionally CaCl2 1 xcexcM and calmodulin 0.1 xcexcM and are added to the reaction batch. The reaction is stopped by addition of 100 xcexcl of acetonitrile which contains 1 mM cAMP and 1 mM AMP. 100 xcexcl of the reaction batch are separated on the HPLC and the cleavage products are determined quantitatively online using a flow-through scintillation counter. The substance concentration at which the reaction rate is decreased by 50% is measured. In addition, the phosphodiesterase [3H] cAMP SPA enzyme assay and the phosphodiesterase [3H] cGMP SPA enzyme assay from Amersham Life Sciences were used for testing. The test was carried out according to the experimental protocol indicated by the manufacturer.
The activity of the test substances on PDE 2 was determined using the [3H] cAMP Scintillation Proximity Assay (SPA) kit (TRKQ7090) from Amersham International (Little Chalfont, England) or on PDE1 and PDE5 using the [3H] cGMP Scintillation Proximity Assay (SPA) Kit (TRKQ7100) from Amersham International (Little Chalfont, England).
Test substances were dissolved in 100% DMSO (10 mM), and this solution was further diluted with H2O (highest final concentration in the test: 10 xcexcM). For the prestimulation of the PDE 2, cGMP is additionally added (final concentration in the test: 10xe2x88x926 M). The enzyme is diluted in PDE buffer (20 mM TRIS/HCl, 5 mM MgCl2, 0.1 mg/ml of albumin, pH 7.5). The following volumes per hole are pipetted into a 96-hole plate (Wallac, 1450-401): 10 xcexcl of substance solution (at the 100% value 10 xcexcl of H2O), 10 xcexcl of cGMP (10xe2x88x925 M), 70 xcexcl of [3H]-cAMP test mixture (see kit), 10 xcexcl of enzyme (at the 0 value no enzyme, instead of this +10 xcexcl of H2O) at the start of the reaction. After incubation at 30xc2x0 C. for 15 min, the reaction was stopped using 50 xcexcl of SPA bead solution (see kit), and the plate was sealed with a film and shaken for 30 seconds. After the beads had settled (about 15 min), the plate was measured in a beta counter.
For the measurement of PDE 1, calmodulin 10xe2x88x927 M and CaCl2 1 xcexcM were added to the reaction batch. The PDE 5 was measured using the [3H] cGMP SPA Assay.
For example, under the conditions indicated above Example 2 inhibits the PDE 2 with an IC50 value of 10 nM.
Measurement of the Increase in the Intracellular Neuronal cGMP Concentration in Cell Cultures
PDE 2 inhibitors increase the intracellular neuronal cGMP concentration after prestimulation of the guanylate cyclase using 10xe2x88x924 M sodium nitroprusside (SNP) in primary rat brain cell cultures.
Rat embryos were decapitated and the heads were transferred to preparation dishes. The scalp and cranium were removed, and the exposed brains were transferred to a further Petri dish. With the aid of a binocular microscope and two pairs of forceps, hippocampi were isolated from the cortex and cooled to 4xc2x0 C. using ice. This preparation and the isolation of the hippocampal neurons were then carried out according to a standard protocol using the papain dissociation system (Worthington Biochemical Corporation, Lakewood, N.J. 08701, USA) (Huettner et al. J. Neurosci. 1986, 6, 3044-3060). The mechanically isolated neurons were cultured under standard conditions (37xc2x0 C., 5% CO2) to 150,000 cells/hole in 200 xcexcl of neurobasal medium/hole (neurobasal; Gibco/BRL; 2 mM L-glutamine; in the presence of penicillin/streptomycin) for 7 days in 96-hole plates (pretreated with poly-D-lysine 100 xcexcg/ml for 20 min). After 7 days, the medium was removed and the cells were washed with HBS buffer (Gibco/BRL). Subsequently, 100 xcexcl each of SNP solution and 100 xcexcl of the racemate of Example 1 (dissolved in 100% DMSO beforehand: 10 mM) were added in HBS to the cells such that the final concentration of SNP was 100 mM and that of the racemate of Example 1 was as indicated in FIG. 1 and the mixture was incubated at 37xc2x0 C. for 20 min. The cells were then lysed in 200 xcexcl of lysis buffer (cGMP kit code RPN 226; from Amersham Pharmacia Biotech.) and the cGMP concentration was measured according to the instructions of the manufacturer. All measurements were carried out in triplicate. Statistical analysis was carried out using Prism Software Version 2.0 (GraphPad Software Inc., San Diego, Calif. USA; *** p less than 0.001).
In the case of parallel incubation of neurons with SNP (a stimulator of guanylate cyclase) and Example 15, a marked increased in the intracellular cGMP level was seen even from a concentration of 100 nM.
Object Recognition Test
The object recognition test is a memory test. It measures the ability of rats (and mice) to differentiate between known and unknown objects and is therefore suitable for the determination of the memory-improving action of the compounds according to the invention.
The test is carried out as described (Blokland et al. NeuroReport 1998, 9, 4205-4208; Ennaceur, A., Delacour, J., Behav. Brain Res. 1988, 31, 47-59; Ennaceur, A., Meliani, K., Psychopharmacology 1992, 109, 321-330; Prickaerts, et al. Eur. J. Pharmacol. 1997, 337, 125-136).
In a first passage, a rat in an otherwise empty relatively large observation arena is confronted with two identical objects. The rat will extensively examine, i.e. sniff and touch, both objects. In a second passage, after an interval of 24 hours, the rat is again tested in the observation arena. One of the known objects is now replaced by a new, unknown object. When a rat recognizes the known object, it will especially examine the unknown object. After 24 hours, a rat, however, has normally forgotten which object it has already examined in the first passage, and will therefore inspect both objects equally intensively. The administration of a substance having learning- and memory-improving action will lead to a rat recognizing the object already seen 24 hours beforehand, in the first passage, as known. It will examine the new, unknown object in greater detail than the already known one. This memory power is expressed in a discrimination index. A discrimination index of zero means that the rat examines both objects, the old and the new one, for the same length of time; i.e. it has not recognized the old object and reacts to both objects as if they were both unknown and new. A discrimination index of greater than zero means that the rat has inspected the new object for longer than the old one; i.e. the rat has recognized the old object.
Under these conditions, Example 16 shows an effect at a dose of 1.0 mg/kg of body weight p.o.
Definitions of Terms
Chromatography, if not mentioned otherwise, was carried out on silica gel Si 60. In the case of flash chromatography, the described conditions were normally followed (cf. Still J. Org. Chem.).
If not described otherwise, the reactions were carried out under argon and, where necessary, under anhydrous conditions.
HPLC=high-pressure liquid chromatography
MS=mass spectrometry
NMR=nuclear magnetic resonance spectroscopy
LC-MS=liquid chromatography combined with mass spectrometry
MeOH=methanol
DMSO=dimethyl sulphoxide
of th.=of theory
Starting Compounds